This invention relates to glutathione transferase (GST) subunits, to nucleic acid sequences encoding glutathione transferase subunits, and to uses of these glutathione transferases and coding sequences, especially in the field of plant biotechnology.
Glutathione transferases (GSTs, EC. 2.5.1.18), also referred to as glutathione S-transferases, are multifunctional enzymes capable of catalysing the conjugation of electrophilic substrates with the tripeptide glutathione (GSH, gamma-glutamylcysteinylglycine). The electrophilic substrate may be of natural or synthetic origin, examples including endogenous stress-metabolites, drugs, pesticides and pollutants. Conjugation with GSH renders the compounds non-toxic and suitable for export from the cytosol and further metabolism. In addition to their activities in GSH conjugation, GSTs may have additional activities as glutathione peroxidases, catalysing the reduction of organic hydroperoxides to the corresponding alcohol according to the reaction:
Rxe2x80x94OOH+2GSHxe2x86x92Rxe2x80x94OH+GSSG.
All known active GSTs are composed of two polypeptide subunits, with each subunit possessing a binding site for GSH and the electrophilic co-substrate. The two subunits may either be identical, giving rise to a homodimer, or dissimilar giving rise to heterodimers. GSTs may therefore be defined according to their source, or class, and their component subunits according to the nomenclature SpGST x-y, where Sp=source or class of GST; x and y describe the subunit types.
Each discrete subunit is encoded by a distinct gene, with many eukaryotes containing GST multigene families encoding multiple isoenzymes.
The plant in which GSTs have been characterised in the greatest detail is maize (Zea mays L.). The major maize GSTs are composed of three discrete subunits, termed I, II and III. These subunits associate together to form three isoenzymes containing the Zea mays GST I subunit, namely ZmGSTI-I, ZmGSTI-II and ZmGSTI-III as well as the homodimers ZmGSTII-II and ZmGSTIII-III. The nucieotide sequences of ZmGSTI, ZmGSTII and ZmGSTIII have been determined. In view of their relatedness in sequence, these maize GSTs have collectively been termed type I plant GSTs. Additional maize GSTs with activities toward herbicides have been described as ZmGSTV-V and ZmGSTV-VI. The sequence of ZmGSTV differs markedly from the other maize GSTs described to date, resembling the auxin-inducible GSTs from dicotyledenous plants which have been termed the type III GSTs.
The maize GST subunit types are associated with differing substrate specificities. The ZmGSTI subunit has broad-ranging, but low, activities toward chloro-s-triazine, chloroacetanilide and diphenyl ether herbicides. The ZmGSTII and ZmGSTIII subunits show greater specificity toward chloroacetanilides, while ZmGSTV and ZmGSTVI are highly active toward diphenyl ethers. The GST isoenzymes differ in their patterns of expression in the organs of maize. Thus, ZmGSTI-I and ZmGSTV-V are expressed in all plant parts, while ZmGSTI-II is root specific. The expression of the GST subunits is also differentially affected by herbicide safeners. These are compounds which enhance the tolerance of cereal crops to herbicides, in part, by increasing the expression of detoxifying enzymes such as GSTs. Thus, the ZmGSTII and ZmGSTV subunits accumulate in maize seedlings following treatment with the safeners dichlormid or benoxacor while the ZmGSTI and ZmGSTIII subunits are only modestly enhanced by safeners.
Far less is known regarding GSTs in plant species other than maize. GSTs with activities toward non-herbicide substrates have been identified in some plants, and mRNAs apparently encoding GSTs have been shown to be expressed in plants including carnation, tobacco and thale cress (Arabidopsis thaliana). However, isoenzymes with activities toward herbicides have only been definitively identified in soybean, pea and pine trees. Of these, only in soybean has the nucleotide coding sequences of the herbicide-detoxifying GST been reported.
GSTs in plants have also been shown to have secondary activities as glutathione peroxidases, able to reduce organic hydroperoxides, such as fatty acid hydroperoxides to the corresponding monohydroxy alcohols. GSTs with glutathione peroxidase activity have been isolated from peas, soybean, A. thaliana and wheat flour. Since fatty acid hydroperoxides are a cornmon result of membrane peroxidation imposed during oxidative stress, glutathione peroxidases provide an important cytoprotective function in preventing the accumulation of fatty acid hydroperoxides and their subsequent degradation to toxic aldehydes. Glutathione peroxidases may therefore have a vital function in protecting plant cells from oxidative stress. The intervention of glutathione peroxidases in lipid peroxidation has also been cited as a determinant of flour quality in wheat.
Of particular relevance to this invention is the lack of knowledge concerning the GSTs of wheat (Triticum aestivum L.).
Some information is available from experiments on whole plants and plant extracts. Several herbicides including examples of the chloroacetanilides, as well as dimethenamid and fenoxaprop-ethyl undergo GSH conjugation in the course of their detoxification in wheat. Also, in crude plant extracts OST activities toward chloroacetanilide herbicides, dimethenamid and fenoxaprop-ethyl have been demonstrated.
There have been very few reports of the purification of GSTs from wheat. A GST was purified from wheat flour, and described as a homodimer of 27.5 kDa polypeptides with activity toward the non-herbicide substrate 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione peroxidase activity toward fatty acid hydroperoxides. A safener-induced GST with activity toward CDNB and dimethenamid, termed GSTTaI-I, has been purified and partially sequenced from the wheat progenitor species Triticum tauschii, (Reichers et al, (1997), Plant Physiology, 114, pages 1461 to 1470).
Moreover, very little is known regarding GST genes in wheat. An mRNA originally described as wir5, which showed sequence similarity to the type 1 maize GSTs, was identified as accumulating in wheat leaves during the onset of acquired resistance to powdery mildew (Erysiphe graminis). The gene was termed gstA1 and shown to be similar in genomic organisation to maize ZmGST1. The gstA1 polypeptide was expressed in recombinant bacteria and shown to have an apparent molecular mass of 29 kDa. The respective enzyme showed GST activity towards the non-herbicide CDNB, though the activity toward other substrates and activity as a glutathione peroxidase was not reported. An antibody was raised to the recombinant GstA1 and used in Western blotting experiments to show that this GST was specifically induced in wheat leaves by pathogen attack. In contrast, a distinct class of GSTs composed of 25 kDa and 26 kDa subunits, which were recognised by an antiserum raised to undefined GSTs in maize, accumulated following exposure to cadmium and the herbicides atrazine, alachlor and paraquat. The activities of these xenobiotic-inducible GSTs in wheat and the corresponding nucleotide sequences were not reported. A cDNA correponding to am mRNA encoding a safener-inducible type III GST has been isolated from Triticum tauschii and had the same amino acid sequence as GSTTaI-I, (Reicher et al, (1997), Plant Physiology, 1141, page 1568).
Thus, although wheat is an important crop plant, there has been little molecular characterisation of wheat GSTs or their genes and, to date, only two purified GSTs and two GST gene sequences, gstA1 and GSTTa1 available.
Significantly, neither purified recombinant GST proteins expressed from gene gstA1 or GSTTa1 were reported to exhibit activity towards herbicides. Hence, none of the previous work on wheat GSTs actually provides any means of achieving herbicide resistance based on the function of wheat GSTs.
We have purified four GST isoenzymes with activity toward herbicides from wheat shoots treated with the herbicide safener fenchlorazole-ethyl and have identified four distinct subunits. In safener-treated shoots, we have found that the predominant GST subunit is a 25 kDa polypeptide, which has been termed Triticum aestivum GST 1 (TaGST1). Additionally, two distinct 26 kDa subunits have been identified and termed TaGST2 and TaGST3 and a 24 kDa subunit, termed TaGST4. These subunits associate together to form the active dimeric isoenzymes TaGST1-1, TaGST1-2, TaGST1-3 and TaGST1-4.
In our experiments, the expression of all four isoenzymes was affected by the herbicide safener fenchlorazole-ethyl, although each one responds in a somewhat different way. The TaGST1-1 isoenzyme is the major GST present in the leaves of untreated wheat seedlings, and its expression is increased by approximately 50% following exposure to fenchlorazole-ethyl. TaGST1-4 is expressed at low levels in untreated shoots and its expression is greatly increased by safener application, while TaGST1-2 and TaGST1-3 are only observed following treatment with the safener. All four of these GST isoenzymes have broad-ranging activities toward xenobiotic substrates and all four demonstrate activity towards herbicides and additional activities as glutathione peroxidases able to reduce organic hydroperoxides, with TaGST1-4 being the most active in this respect. Each isoenzyme also has specific properties. Thus, for example, detoxification of one particular herbicide, fenoxaprop-ethyl, is associated with the more strongly safener-inducible TaGST1-2, TaGST1-3 and TaGST1-4 heterodimers, rather than with the TaGST1-1 homodimer.
Furthermore, we have identified, cloned and sequenced cDNAs for the major type III GSTs in wheat, together with cDNAs encoding a range of type I GSTs, all active in herbicide metabolism. This is fundamental to understanding the GST detoxification system in wheat and to exploiting it to generate transgenic herbicide-resistant plants expressing wheat GSTs. In many previous studies, GST activity could not be linked to specific genes, precluding this approach.
From the sequences of the cDNAs the amino acid sequences of the GST subunits themselves has been deduced.
Accordingly, the invention provides:
a polynucleotide encoding a glutathione transferase (GST) subunit, which polynucleotide comprises a coding sequence capable of hybridising selectively to the coding sequence of SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15 or 17 to the complement of one of those sequences.
The invention also provides:
a polypeptide which is a GST subunit and comprises the amino acid sequence of SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16 or 18 or a sequence substantially homologous thereto, or a fragment of either said sequence.
The invention also provides:
a dimeric protein comprising two GST subunits, wherein at least one subunit is a polypeptide of the invention.
The invention also provides:
a chimeric gene comprising a polynucleotide of the invention operably linked to regulatory sequences that allow expression of the coding sequence in a host cell.
The invention also provides:
a vector comprising a polynucleotide of the invention or a chimeric gene of the invention.
The invention also provides:
a cell transformed or transfected with a vector of the invention.
The invention also provides:
a cell having, integrated into its genome, a chimeric gene of the invention.
The invention also provides:
a process for the production of a polypeptide of the invention, which process comprises:
(a) cultivating a cell of the invention under conditions that allow the expression of the polypeptide; and
(b) recovering the expressed polypeptide.
The invention also provides:
a process for the production of a dimeric protein of the invention, which process comprises:
(a) cultivating a cell of the invention under conditions that allow:
(i) the expression of the polypeptide of the invention and, if a further polynucleotide sequence as defined herein is present, optionally the expression of a further GST subunit encoded by a further polynucleotide, and
(ii) the association of the GST subunit polypeptide of the invention with another GST subunit polypeptide to form a dimeric protein of the invention; and
(b) recovering the dimeric protein so formed.
The invention also provides:
a method of obtaining a transgenic plant cell comprising:
(a) transforming a plant cell with an expression vector of the invention to give a transgenic plant cell,
and optionally,
(axe2x80x2) transforming the cell with one or more further polynucleotide sequences coding for a GST subunit, operably linked to regulatory elements that allow expression of the subunit in the cell.
The invention also provides:
a method of obtaining a first-generation transgenic plant comprising:
(b) regenerating a transgenic plant cell transformed with a vector of the invention to give a transgenic plant.
The invention also provides:
a method of obtaining a transgenic plant seed comprising:
(c) obtaining a transgenic seed from a transgenic plant obtainable by regenerating a transgenic plant cell transformed with a vector of the invention.
The invention also provides:
a method of obtaining a transgenic progeny plant comprising obtaining a second-generation transgenic progeny plant from a first-generation transgenic plant obtainable by regenerating a transgenic plant cell transformed with a vector of the invention, and optionally obtaining transgenic plants of one or more further generations from the second-generation progeny plant thus obtained.
The invention also provides:
a method of obtaining a transgenic progeny plant comprising obtaining a second-generation transgenic progeny plant from a first-generation transgenic plant obtainable by regenerating a transgenic plant cell transformed with a vector of the invention comprising:
(c) obtaining a transgenic seed from a first-generation transgenic plant obtainable by regenerating a transgenic plant cell transformed with a vector of the invention, then obtaining a second-generation transgenic progeny plant from the transgenic seed;
and/or
(d) propagating clonally a first-generation transgenic plant obtainable by regenerating a transgenic plant cell transformed with a vector of the invention to give a second-generation progeny plant;
and/or
(e) crossing a first-generation transgenic plant obtainable by regenerating a transgenic plant cell transformed with a vector of the invention with another plant to give a second-generation progeny plant;
and optionally;
(f) obtaining transgenic progeny plants of one or more further generations from the second-generation progeny plant thus obtained.
The invention also provides:
a transgenic plant cell, first-generation plant, plant seed or progeny plant obtainable by a method of the invention.
The invention also provides:
a transgenic plant or plant seed comprising plant cells of the invention.
The invention also provides:
a transgenic plant cell callus comprising plant cells of the invention, or obtainable from a transgenic plant cell, first-generation plant, plant seed or progeny plant of the invention.
The invention also provides:
use of a polynucleotide of the invention as a selectable marker for detecting transformation of a plant cell.
The invention also provides:
a nucleic acid construct comprising:
(a) a polynucleotide of the invention operably linked to regulatory elements that allow expression of the coding sequence in a plant cell; and
(b) a site into which a further polynucleotide comprising a coding sequence can be inserted.
The invention also provides:
a vector comprising such a construct.
The invention also provides:
a method of transforming a plant cell or of obtaining a plant cell culture or transgenic plant comprising:
(a) providing an untransformed plant cell which is susceptible to a herbicide whose herbicidal activity is reduced by a dimeric protein of the invention;
(b) transforming the plant cell with a vector comprising:
(i) a polynucleotide of the invention operably linked to regulatory elements that allow expression of the coding sequence in a plant cell; and
(ii) a site into which a further polynucleotide comprising a coding sequence can be inserted;
(c) cultivating the transformed cell under conditions that allow the expression of the polynucleotide (a) in the construct; and/or
(cxe2x80x2) regenerating the cell to give a cell culture or plant such that the polynucleotide (a) in the construct is expressed; and
(d) contacting the cell, cell culture or plant with the herbicide whose herbicidal activity is reduced by the dimeric protein of the invention, and to which the untransformed plant cell was susceptible; and
(e) selecting cells, cell cultures or plants that are less susceptible to the herbicide than are corresponding untransformed cells, cell cultures or plants.
The invention also provides:
use of a dimeric protein of the invention in a method of identifying compounds capable of metabolism by a GST.
The invention also provides:
a method of identifying compounds capable of being metabolised by a glutathione transferase comprising:
(a) contacting a candidate compound suspected of being capable of being metabolised by glutathione transferase with glutathione (GSH) in the presence of a dimeric protein of the invention; and
(b) determining whether or not metabolism of the candidate compound takes place.
The invention also provides:
compounds identified by such methods.
The invention also provides:
a kit for detecting compounds capable of being metabolised by a GST comprising:
(a) reduced glutathione, hydroxymethylglutathione or homoglutathione;
and
(b) a dimeric protein of the invention.
The invention also provides:
an antibody which specifically recognises a polypeptide or dimeric protein of the invention.
The invention also provides:
a nucleic acid probe which selectively hybridises to the sequence of SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15 or 17.
The invention also provides:
a method of identifying compounds that induce GST expression in graminaceous plants comprising:
(a) contacting a graminaceous plant, or a cell or cell culture thereof, with a candidate compound suspected of being capable of inducing GST expression; and
(b) determining the level of GST expression in the plant, cell or cell culture.
The invention also provides:
compounds identified by such methods.
The invention also provides:
a kit for identifying compounds that induce GST expression in plants by such a method, which kit comprises an antibody of the invention.
The invention also provides:
a method of determining the GST level in a sample of seed or flour comprising:
(i) determining the level of GST protein present by using an antibody of the invention; or
(ii) determining the level of GST mRNA present using a probe of the invention.
The invention also provides:
a method of controlling the growth of weeds at a locus where a transgenic plant of the invention is being cultivated, which method comprises applying to the locus a herbicide whose herbicidal properties are reduced by a dimeric protein of the invention.